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To investigate the regulatory effect and mechanism of vitamin D on the local renin-angiotensin system at maternal-fetal interface in the pathological process of preeclampsia (PE).The mRNA and protein expression of renin in decidua of normal pregnancy and PE placentas was determined by RT-PCR and Western blot. Normal decidual tissues were treated with active and inactive vitamin D for 48 hThe expression of renin in PE decidua was significantly higher than that of normal control at transcriptional and translational levels (The expression of renin in placental decidua is up-regulated in patients with PE, and the activation of local renin-angiotensin system at the maternal-fetal interface may be involved in the pathogenesis of PE. Vitamin D can specifically down-regulate renin expression in human decidual gland epithelial cells by competing with the PKA pathway. Vitamin D supplementation may have potential value for clinical intervention of PE.

Background Ocular blood flow provides a new perspective for studying the effects of diabetes and ischemic heart disease on systemic blood flow, pathological mechanisms, and prognosis. Previous studies have analyzed the hemodynamic changes of the ophthalmic artery (OA) in patients with diabetes and ischemic heart disease, but the results remain controversial due to limited observation methods. We aimed to explore the morphological and hemodynamic features in the OA in patients with type 2 diabetes (T2D) with and without acute coronary syndrome (ACS). Methods In total, 134 participants, including 30 control participants, 34 with ACS only, 34 with T2D only, and 36 with both ACS and T2D, undergoing computed tomography angiography were enrolled. Three-dimensional OA models were reconstructed, and morphological parameters of the OA were measured. In addition, numerical simulations using computational fluid dynamics were used to acquire hemodynamic parameters of the OA. Results In this study, 134 OA models were reconstructed. Morphological measurements revealed a smaller initial OA diameter in the T2D group than in the other two ACS groups. A hemodynamic simulation showed a significantly lower OA blood velocity in patients with ACS and T2D than that in controls (P P P = 0.020, and P P = 0.013). The OA blood velocity and mass flow ratio were correlated with several clinical parameters. Conclusions This study revealed morphological and hemodynamic differences in the OA between patients with T2D with and without ACS. Furthermore, the hemodynamic characteristics of the OA correlated with clinical prognostic biomarkers, suggesting the potential predictive ability of the OA.

Successful pregnancy in placental mammals substantially depends on the establishment of maternal immune tolerance to the semi-allogenic fetus. Disorders in this process are tightly associated with adverse pregnancy outcomes including recurrent miscarriage (RM). However, an in-depth understanding of the systematic and decidual immune environment in RM remains largely lacking. In this study, we utilized single-cell RNA-sequencing (scRNA-seq) to comparably analyze the cellular and molecular signatures of decidual and peripheral leukocytes in normal and unexplained RM pregnancies at the early stage of gestation. Integrative analysis identifies 22 distinct cell clusters in total, and a dramatic difference in leukocyte subsets and molecular properties in RM cases is revealed. Specifically, the cytotoxic properties of CD8+ effector T cells, nature killer (NK), and mucosal-associated invariant T (MAIT) cells in peripheral blood indicates apparently enhanced pro-inflammatory status, and the population proportions and ligand–receptor interactions of the decidual leukocyte subsets demonstrate preferential immune activation in RM patients. The molecular features, spatial distribution, and the developmental trajectories of five decidual NK (dNK) subsets have been elaborately illustrated. In RM patients, a dNK subset that supports embryonic growth is diminished in proportion, while the ratio of another dNK subset with cytotoxic and immune-active signature is significantly increased. Notably, a unique pro-inflammatory CD56+CD16+ dNK subset substantially accumulates in RM decidua. These findings reveal a comprehensive cellular and molecular atlas of decidual and peripheral leukocytes in human early pregnancy and provide an in-depth insight into the immune pathogenesis for early pregnancy loss.

Background Preeclampsia (PE), a placenta-associated pregnancy complication, is the leading cause of maternal and perinatal morbidity and mortality. Met/Erk signaling is inhibited in the placentas of patients with early-onset preeclampsia (E-PE), but the underlying mechanisms remain elusive. In this study, the expression modes of Met and endocytic vesicles in normal and preeclamptic placentas were compared. Biotinylation internalization/recycling assays were used to measure the endocytosis of Met under hypoxia and normoxia in HTR8/SVneo cells. In addition, the expression level of Cbl, a specific E3 ligase of Met, was measured under hypoxia and normoxia, and the endocytosis of Met was studied by using confocal microscopy. Results We found considerable intracellular accumulation of Met, which was colocalized with caveolin-1 (CAV-1), in trophoblasts from E-PE placentas. Prolonged hypoxic stimulation led to the remarkable augmentation of CAV-1-mediated Met endocytosis in HTR8/SVneo cells. In addition, the expression of Cbl was substantially repressed by sustained hypoxia, disrupting ubiquitin degradation and the subsequent intracellular accumulation of Met in HTR8/SVneo cells. The abnormal degradation of Met hampered the ability of hepatocyte growth factor (HGF) to promote trophoblast cell invasion. In E-PE placentas, aberrant upregulation of CAV-1 and downregulation of Cbl were observed in parallel to the intracellular accumulation of Met. Conclusions These findings reveal that prolonged hypoxic stress induces the augmentation of endocytosis and repression of ubiquitin-mediated Met degradation, which leads to the impaired regulation of trophoblast invasion by HGF/Met signaling. These data provide novel evidence for elucidating the pathogenesis of preeclampsia, especially of the early-onset subtype.

The impacts of the current COVID-19 pandemic on maternal and foetal health are enormous and of serious concern. However, the influence of SARS-CoV-2 infection at early-to-mid gestation on maternal and foetal health remains unclear.Here, we report the follow-up study of a pregnant woman of her whole infective course of SARS-CoV-2, from asymptomatic infection at gestational week 20 to mild and then severe illness state, and finally cured at Week 24. Following caesarean section due to incomplete uterine rupture at Week 28, histological examinations on the placenta and foetal tissues as well as single-cell RNA sequencing (scRNA-seq) for the placenta were performed.Compared with the gestational age-matched control placentas, the placenta from this COVID-19 case exhibited more syncytial knots and lowered expression of syncytiotrophoblast-related genes. The scRNA-seq analysis demonstrated impaired trophoblast differentiation, activation of antiviral and inflammatory CD8 T cells, as well as the tight association of increased inflammatory responses in the placenta with complement over-activation in macrophages. In addition, levels of several inflammatory factors increased in the placenta and foetal blood.These findings illustrate a systematic cellular and molecular signature of placental insufficiency and immune activation at the maternal-foetal interface that may be attributed to SARS-CoV-2 infection at the midgestation stage, which highly suggests the extensive care for maternal and foetal outcomes in pregnant women suffering from COVID-19.

Purpose To examine the morphological and hemodynamic changes of the ophthalmic artery (OA) in patients with acute coronary syndrome (ACS). Methods This cross-sectional observational study included 31 patients with ACS and 10 healthy controls (HCs). The ACS subgroups were ST-segment elevation myocardial infarction (STEMI; n = 10), non-STEMI (n = 10), and unstable angina (n = 11). OA three-dimensional (3D) models were reconstructed based on computed tomographic angiography, and morphological aspects of the OA were measured quantitatively. Moreover, numerical simulation by computational fluid dynamics was used to obtain hemodynamic information of the OA. Results The study reconstructed 41 OA models. Hemodynamic simulation revealed a significant decrease in OA blood velocity in patients with ACS compared with the HCs (median velocity, 0.046 vs. 0.147 m/s; P < 0.001). No differences in the morphological data for the OA were observed. Also, no differences in the mass flow ratio of OA to the ipsilateral internal carotid artery was found. Similar differences were observed between the ACS subgroups and HCs. OA blood velocity was negatively correlated with body mass index, abdominal circumference, left ventricular ejection fraction, and triacylglycerol and was positively correlated with early to late transmitral flow velocity, N-terminal pro-brain natriuretic peptide, serum creatinine, and potassium. Conclusions The initial OA blood velocity was slower in patients with ACS and was associated with ACS-related clinical parameters. To our knowledge, this is the first study to analyze OA characteristics in ACS using 3D model reconstruction and hemodynamic simulation, providing new perspectives on the relationship between ischemic heart disease and ocular manifestations.

Objectives Success in pregnancy in mammals predominantly depends on a well‐developed placenta. The differentiation of invasive trophoblasts is a fundamental process of placentation, the abnormalities of which are tightly associated with pregnancy disorders including preeclampsia (PE). Monoclonal nonspecific suppressor factor beta (MNSFβ) is an immunosuppressive factor. Its conventional knockout in mice induced embryonic lethality, whereas the underlying mechanism of MNSFβ in regulating placentation and pregnancy maintenance remains to be elucidated. Methods Trophoblast‐specific knockout of MNSFβ was generated using Cyp19‐Cre mice. In situ hybridization (ISH), haematoxylin and eosin (HE), immunohistochemistry (IHC) and immunofluorescence (IF) were performed to examine the distribution of MNSFβ and insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) at the foeto‐maternal interface. The interaction and expression of MNSFβ, IGF2BP2 and invasion‐related molecules were detected by immunoprecipitation (IP), immunoblotting and quantitative real‐time polymerase chain reaction (qRT‐PCR). The cell invasion ability was measured by the Transwell insert assay. Results We found that deficiency of MNSFβ in trophoblasts led to embryonic growth retardation by mid‐gestation and subsequent foetal loss, primarily shown as apparently limited trophoblast invasion. In vitro experiments in human trophoblasts demonstrated that the conjugation of MNSFβ with IGF2BP2 and thus the stabilization of IGF2BP2 essentially mediated the invasion‐promoting effect of MNSFβ. In the placentas from MNSFβ‐deficient mice and severe preeclamptic (PE) patients, downregulation of MNSFβ was evidently associated with the repressed IGF2BP2 expression. Conclusions The findings reveal the crucial role of MNSFβ in governing the trophoblast invasion and therefore foetal development, and add novel hints to reveal the placental pathology of PE., In placental mammals, the appropriate differentiation of placental trophoblast cell along the invasive pathway predominantly governs placentation and reproductive success. Here, we reveal the crucial role of MNSFβ in governing trophoblast cell invasion via interaction with IGF2BP2. Attenuated expression of placental MNSFβ and IGF2BP2 leads to limited trophoblastic invasiveness and pregnancy disorder including preeclampsia (PE).

Background To investigate the structural and functional changes of the retina in patients with different degrees of internal carotid artery (ICA) stenosis. Methods This cross-sectional study included patients with varying degrees ICA stenosis. Clinical characteristics of 41 patients were collected after being divided into four groups according to the ICA stenosis indicated by computed tomographic angiography (Group 0: without ICA stenosis, Group 1: ipsilateral slight ICA stenosis, Group 2: ipsilateral moderate ICA stenosis, Group 3: ipsilateral severe ICA stenosis). Retinal vessel caliber (RVC) was measured quantitatively with the Integrative Vessel Analysis software. The retinal sensitivity was examined with the MP-3 microperimeter. The relationships among central retinal artery equivalent (CRAE), central retinal vein equivalent, arteriole to venule ratio (AVR), mean retinal sensitivity (MS) and ICA stenosis degree were analysed. Results The CRAE in Group 3 were significantly smaller compared with Group 0, Group 1 and Group 2 (P P P = 0.002). Significant decrease was found between Group 3 with other groups in MS at fovea (P P P = 0.002). Moreover, there was a positive correlation found between MS and CRAE (Beta = 0.60, P P P P Conclusions The present study revealed smaller CRAE and AVR in ipsilateral severe ICA stenosis patients. And the MS decreased in patients with severe ICA stenosis. In addition, MS had a positive correlation with CRAE.

Decidual macrophages (dMϕ) are the second largest population of leukocytes at the maternal–fetal interface and play critical roles in maintaining pregnancy. Our previous studies demonstrated the active involvement of monoclonal nonspecific suppressor factor-β (MNSFβ) in embryonic implantation and pregnancy success. MNSFβ is a ubiquitously expressed ubiquitin-like protein that also exhibits immune regulatory potential, but its function in human dMϕ remains unknown. Here, we observed that the proportion of CD11chigh(CD11cHI) dMϕ was significantly increased in dMϕ derived from patients with recurrent pregnancy loss (RPL dMϕ) compared to those derived from normal pregnant women (Control dMϕ). The production of MNSFβ and TNFα by RPL dMϕ was also significantly increased compared to that by Control dMϕ. Conditioned medium from RPL dMϕ exerted an inhibitory effect on the invasiveness of human trophoblastic HTR8/SVneo cells, and this effect could be partially reversed by a neutralizing antibody against TNFα. Bioinformatics analysis indicated a potential interaction between MNSFβ and RC3H1, a suppressor of TNFα transcription. Immunoprecipitation experiments with human Mϕ differentiated from the human monocyte cell line Thp1 (Thp1-derived Mϕ) proved the binding of MNSFβ to RC3H1. Specific knockdown of MNSFβ in Thp1-derived Mϕ led to a marked decrease in TNFα production, which could be reversed by inhibiting RC3H1 expression. Interestingly, a significant decrease in the protein level of RC3H1 was observed in RPL dMϕ. Together, our findings indicate that aberrantly increased MNSFβ expression in dMϕ may promote TNFα productionviaits interaction with RC3H1, and these phenomena could result in the disruption of the immune balance at the maternal–fetal interface and thus pregnancy loss.

Itaconate has been recently recognized as an anti-inflammatory metabolite involved in the pathogen-macrophage interface. Due to its weak electrophilicity, itaconate could modify cysteines of the protein KEAP1 and glutathione, which contribute to its anti-inflammatory effect. However, the substrates of itaconate modification in macrophages have not been systematically profiled, which largely impedes the understanding of its roles in immune responses. Here, we developed a specific thiol-reactive probe, 1-OH-Az, for quantitative chemoproteomic profiling of cysteine modifications by itaconate, and provided a global portrait of its proteome reactivity. We found that itaconate covalently modifies key glycolytic enzymes and impairs glycolytic flux mainly through inhibition of fructose-bisphosphate aldolase A (ALDOA). Moreover, itaconate attenuates the inflammatory response in stimulated macrophages by impairing the glycolysis. Our study provides a valuable resource of protein targets of itaconate in macrophages and establishes a negative-feedback link between glycolysis and itaconate, elucidating new functional insights for this anti-inflammatory metabolite.

During pregnancy, the appropriate allocation of nutrients between the mother and the fetus is dominated by maternal–fetal interactions, which is primarily governed by the placenta. The syncytiotrophoblast (STB) lining at the outer surface of the placental villi is directly bathed in maternal blood and controls feto–maternal exchange. The STB is the largest multinucleated cell type in the human body, and is formed through syncytialization of the mononucleated cytotrophoblast. However, the physiological advantage of forming such an extensively multinucleated cellular structure remains poorly understood. Here, we discover that the STB uniquely adapts to nutrient stress by inducing the macropinocytosis machinery through repression of mammalian target of rapamycin (mTOR) signaling. In primary human trophoblasts and in trophoblast cell lines, differentiation toward a syncytium triggers macropinocytosis, which is greatly enhanced during amino acid shortage, induced by inhibiting mTOR signaling. Moreover, inhibiting mTOR in pregnant mice markedly stimulates macropinocytosis in the syncytium. Blocking macropinocytosis worsens the phenotypes of fetal growth restriction caused by mTOR-inhibition. Consistently, placentas derived from fetal growth restriction patients display: 1) Repressed mTOR signaling, 2) increased syncytialization, and 3) enhanced macropinocytosis. Together, our findings suggest that the unique ability of STB to undergo macropinocytosis serves as an essential adaptation to the cellular nutrient status, and support fetal survival and growth under nutrient deprivation.

Region-related heterogeneity and state transitions of microglia are important for brain development and neurological pathogenesis. However, regional specialization and state transition in microglia during early human CNS development remain unclear. Here, we profile single-cell transcriptomes of microglia from distinct regions of the developing human brain, and combined with experimental verification, we define and characterize early microglial fate determinations related to regional specification and state transition. We identified several subclasses of neuronal gene-enriched microglia with regional specification that dynamically and transiently appeared as early brain regions formed. In contrast, immune-related microglia were regionally specialized at later stages of CNS development. Surprisingly, we discovered that region-specialized immune-related microglia exit from a relative resting state and transition into distinct active states. In addition, we experimentally verified the microglial state transition. Finally, we showed that the state transition is conserved but that there are molecular differences in developing microglia in humans and mice.

Uterine spiral artery (SPA) remodeling is a crucial event during pregnancy to provide enough blood supply to maternal–fetal interface and meet the demands of the growing fetus. Along this process, the dynamic change and the fate of spiral artery vascular smooth muscle cells (SPA–VSMCs) have long been debatable. In the present study, we analyzed the cell features of SPA–VSMCs at different stages of vascular remodeling in human early pregnancy, and we demonstrated the progressively morphological change of SPA–VSMCs at un-remodeled (Un-Rem), remodeling, and fully remodeled (Fully-Rem) stages, indicating the extravillous trophoblast (EVT)-independent and EVT-dependent phases of SPA–VSMC dedifferentiation. In vitro experiments in VSMC cell line revealed the efficient roles of decidual stromal cells, decidual natural killer cells (dNK), decidual macrophages, and EVTs in inducing VSMCs dedifferentiation. Importantly, the potential transformation of VSMC toward CD56+ dNKs was displayed by immunofluorescence-DNA in-situ hybridization-proximity ligation and chromatin immunoprecipitation assays for H3K4dime modification in the myosin heavy chain 11 (MYH11) promoter region. The findings clearly illustrate a cascade regulation of the progressive dedifferentiation of SPA–VSMCs by multiple cell types in uterine decidual niche and provide new evidences to reveal the destination of SPA–VSMCs during vascular remodeling.

Placenta serves as a temporary fetal organ, which mediates maternal-fetal crosstalk and intrauterine fetal growth. Placental defensive barrier is a fundamental physiological function, which balances maternal immune tolerance to the fetus and resistance to pathogens. This review summarizes the latest research progress on the mechanisms of placental barrier formation from the view of placental development. Recent discoveries have shed light on the cellular and molecular properties of placental defensive mechanisms in syncytiotrophoblast, including autophagy, exosome mediated anti-pathogenic pathways, cell-cell junctions and cytoskeleton networks. We also present an overview of placental barrier dysfunction and its implications in intrauterine TORCH infections.

Objectives During human pregnancy, the endothelial cells of the uterine spiral arteries (SPA) are extensively replaced by a subtype of placental trophoblasts, endovascular extravillous trophoblasts (enEVTs), thus establishing a placental‐maternal circulation. On this pathway, foetus‐derived placental villi and enEVTs bath into the maternal blood that perfuses along SPA being not attacked by the maternal lymphocytes. We aimed to reveal the underlying mechanism of such immune tolerance. Methods In situ hybridization, immunofluorescence, ELISA and FCM assay were performed to examine TGF‐β1 expression and distribution of regulatory T cells (Tregs) along the placental‐maternal circulation route. The primary enEVTs, interstitial extravillous trophoblasts (iEVTs) and decidual endothelial cells (dECs) were purified by FACS, and their conditioned media were collected to treat naïve CD4+ T cells. Treg differentiation was measured by FLOW and CFSE assays. Results We found that enEVTs but not iEVTs or dECs actively produced TGF‐β1. The primary enEVTs significantly promoted naïve CD4+ T‐cell differentiation into immunosuppressive FOXP3+ Tregs, and this effect was dependent on TGF‐β1. In recurrent spontaneous abortion (RSA) patients, an evidently reduced proportion of TGF‐β1–producing enEVTs and their ability to educate Tregs differentiation were observed. Conclusions Our findings demonstrate a unique immune‐regulatory characteristic of placental enEVTs to develop immune tolerance along the placental‐maternal circulation. New insights into the pathogenesis of RSA are also suggested.

Objective The signalling of the receptor tyrosine kinase Met is critical in promoting trophoblast cell invasion, and the deficiency in HGF/Met signalling is associated with preeclampsia. The semaphorin family member semaphorin4D (sema4D) and its receptor Plexin-B1 have been reported to control tumour cell invasion by coupling with Met. We hypothesized that sema4D/Plexin-B1 may promote trophoblast invasion by activating Met, and downregulation of sema4D/Plexin-B1 may account for the deficiency in Met signalling in preeclamptic placenta. Methods In this study, Met and Erk activation and the expression of sema4D/Plexin-B1 in normal and preeclamptic placentas were comparably measured. The role of sema4D in trophoblast cell invasion and tubulogenesis was examined in vitro using the Transwell invasion assay and tube formation assay in trophoblast-endothelial cell co-culture model. Results Met, sema4D and Plexin-B1 co-localized in various subtypes of human trophoblast cells, including villous trophoblasts and extravillous trophoblasts (EVTs). In early-onset preeclampsia (E-PE) placentas, the phosphorylated Met and Erk as well as sema4D and Plexin-B1 were much lower than those in gestational week-matched preterm-labour (PTL) placentas. In human trophoblast HTR8/SVneo cell line, sema4D could promote Met and Erk phosphorylation as well as enhance trophoblast cell invasion and tubulogenesis with endothelial cells. Moreover, the effect of sema4D on HTR8/SVneo could be blocked by knocking down Met with specific siRNA. Conclusion The crosstalk between sema4D and Met could transactivate Met to promote trophoblast cell invasion and differentiation, and decreased expression of sema4D and Plexin-B1 may be responsible for the deficiency in Met signalling and the development of preeclampsia.

Preeclampsia (PE) is the leading cause of maternal and neonatal morbidity and mortality. Defects in trophoblast invasion, differentiation of endovascular extravillous trophoblasts (enEVTs), and spiral artery remodeling are key factors in PE development. There are no markers clinically available to predict PE, leaving expedited delivery as the only effective therapy. Dysregulation of miRNA in clinical tissues and maternal circulation have opened a new avenue for biomarker discovery. In this study, we investigated the role of miR-218-5p in PE development. miR-218-5p was highly expressed in EVTs and significantly downregulated in PE placentas. Using first-trimester trophoblast cell lines and human placental explants, we found that miR-218-5p overexpression promoted, whereas anti-miR-218-5p suppressed, trophoblast invasion, EVT outgrowth, and enEVT differentiation. Furthermore, miR-218-5p accelerated spiral artery remodeling in a decidua-placenta co-culture. The effect of miR-218-5p was mediated by the suppression of transforming growth factor (TGF)-β2 signaling. Silencing of TGFB2 mimicked, whereas treatment with TGF-β2 partially reversed, the effects of miR-218-5p. Taken together, these findings demonstrate that miR-218-5p promotes trophoblast invasion and enEVT differentiation through a novel miR-218-5p-TGF-β2 pathway. This study elucidates the role of an miRNA in enEVT differentiation and spiral artery remodeling and suggests that downregulation of miR-218-5p contributes to PE development.

Decrease of chloride concentration contributes to cardiovascular diseases, however, whether decrease of chloride concentration is involved in platelet activation remains elusive. In the present study, we found that ACI patients had lower serum chloride which would be rescued after Aspirin administration. ADP induced chloride concentration reduction in platelets. Blockade of chloride channel prevented ADP-induced platelet adhesion, activation and aggregation, however, decreasing the extracellular chloride concentration promoted ADP-induced platelet adhesion and activation. Decrease of the extracellular chloride concentration facilitated the inactivation of Src family kinase Lyn, which was not involved in PI3K/Akt phosphorylation. Nevertheless, low chloride concentration promoted the production of platelet cytosol Gαi2 subunit. This subunit prevents AC from converting ATP into cAMP, which therefore, inhibited the phosphorylation of PKA to promote platelet activation. In conclusion, decreased intracellular chloride promotes ADP induced platelet activation through the Gαi2/cAMP/PKA pathway instead of the Lyn/PI3K/Akt signal pathway.

BMP2 expression is spatiotemporally correlated with embryo implantation and is crucial for endometrial decidualization and fertility in mice. BMP2 has been reported to increase the mesenchymal adhesion molecule N-cadherin and enhance cell invasion in cancer cells; moreover, studies suggest that N-cadherin promotes placental trophoblast invasion. However, whether BMP2 can promote trophoblast cell invasion during placentation remains unknown. The objective of our study was to investigate the effects of BMP2 on human trophoblast cell invasion and the involvement of N-cadherin and SMAD signaling. Primary and immortalized (HTR8/SVneo) cultures of human extravillous trophoblast (EVT) cells were used as study models. Treatment with recombinant human BMP2 increased HTR8/SVneo cell transwell Matrigel invasion as well as N-cadherin mRNA and protein levels, but had no significant effect on cell proliferation. Likewise, BMP2 treatment enhanced primary human EVT cell invasion and N-cadherin production. Basal and BMP2-induced invasion were attenuated by small interfering RNA-mediated downregulation of N-cadherin in both HTR8/SVneo and primary EVT cells. Intriguingly, BMP2 induced the phosphorylation/activation of both canonical SMAD1/5/8 and non-canonical SMAD2/3 signaling in HTR8/SVneo and primary EVT cells. Knockdown of SMAD2/3 or common SMAD4 totally abolished the effects of BMP2 on N-cadherin upregulation in HTR8/SVneo cells. Upregulation of SMAD2/3 phosphorylation and N-cadherin were totally abolished by type I receptor activin receptor-like kinases 2/3 (ALK2/3) inhibitor DMH1; moreover, knockdown of ALK2 or ALK3 inhibited N-cadherin upregulation. Interestingly, activation of SMAD2/3 and upregulation of N-cadherin were partially attenuated by ALK4/5/7 inhibitor SB431542 or knockdown of ALK4, but not ALK5. Our results show that BMP2 promotes trophoblast cell invasion by upregulating N-cadherin via non-canonical ALK2/3/4-SMAD2/3-SMAD4 signaling.

Decidual NK (dNK) cells, identified as CD56brightCD16-CD3-, account for ~70% of lymphocytes within the uterine wall during early pregnancy. Accumulating evidence suggests that tight interactions between placental trophoblasts and dNK cells are critical for trophoblast cell differentiation. However, the underlying mechanism remains to be explored in detail. In the present study, conditioned medium (CM) was collected from cultured primary human dNK cells. Primary cytotrophoblasts (CTBs) or the human trophoblast cell line HTR8/SVneo was treated with dNK-CM and co-cultured with human umbilical vein endothelial cells (HUVECs) in a three-dimensional Matrigel scaffold, and the formation of tube structures was dynamically monitored with live cell imaging. Trophoblast invasion was analyzed with a transwell invasion assay. The data demonstrated that the treatment of HTR8/SVneo cells or CTBs with dNK-CM remarkably promoted trophoblast invasion and tube formation in the presence of HUVECs. The epithelial marker E-cadherin was reduced, while the expression of endothelial markers NCAM, VE-cadherin and integrin β1 was significantly promoted in the HTR8/SVneo cells upon treatment with dNK-CM. Antibody blocking experiments revealed that the dNK cells promoted trophoblast invasion through the production of IL-8 and HGF, and they induced trophoblast differentiation toward endothelial phenotype by producing VEGF-C and HGF. These results provide new evidence to clarify the finely tuned interactions between trophoblasts and dNK cells at the maternal-fetal interface.

This study aimed to measure the basal energy expenditure (BEE) of Chinese healthy adults and establish an accurate predictive equation for this population.In total, 470 Chinese healthy adults had their BEE measured using the Cosmed K4bThere was a significant difference in BEE between males and females, with 5,954 kJ/d and 5,089 kJ/d, respectively. People living in rural areas expended significantly higher BEE (5,885 kJ/d) than those in urban areas (5,279 kJ/d). Previous equations developed by Henry, Schofield, Harris-Benedict (H-B), and Liu overestimated the BEE of Chinese healthy adults. The new equations derived from the present study displayed the smallest average bias and RMSE from the measured basal energy expenditure (mBEE). The CCC of the new equations was higher than other predictive equations, but it was lower than 0.8. There was no significant difference in the accuracy rate among all predictive equations.Sex and regional differences in BEE were observed in Chinese healthy adults. Neither the widely used previous predictive equations nor the one derived in the present study were accurate enough for estimating the BEE of Chinese healthy adults. Further study is required to develop more accurate equations for predicting the BEE of Chinese healthy adults aged between 20-45 years.

The pregnancy complication preeclampsia is directly associated with hypoxic stress and insufficient trophoblast cell differentiation. The hypoxia-inducible microRNA (miRNA), miR-210, has been identified as a significantly up-regulated miRNA in preeclamptic placenta, and evidence in other cell types has indicated a feedback regulation between miR-210 and hypoxia-inducible factor-1α (HIF-1α) under hypoxic condition. It remains unclear whether and how the feedback loop between miR-210 and HIF-1α may contribute to trophoblast dysfunction in preeclampsia. Here we proved that cytoplasmic polyadenylation element-binding 2 (CPEB2) was a direct target of miR-210 in human trophoblast. CPEB2 could inhibit the translation of hypoxia-induced HIF-1α via directly binding the cytoplasmic polyadenylation element (CPE) site in the 3′-untranslated region (UTR) of HIF-1α mRNA. The increase in the HIF-1α level upon hypoxia treatment could be efficiently reversed by miR-210 inhibitor. In addition, CPEB2 was primarily expressed in villous syncytiotrophoblasts, and the suppression of trophoblast cell syncytialization by miR-210 could be significantly rescued by CPEB2 overexpression. In preeclamptic placenta, the expression of CPEB2 was evidently lower than normal pregnant control, and the miR-210 level was aberrantly higher and trophoblast syncytialization was limited. The findings revealed a positive feedback loop between miR-210 and HIF-1α that is mediated by CPEB2 in human trophoblasts, and demonstrated a mechanism underlying the insufficient trophoblast syncytialization in preeclampsia under hypoxic stress.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder associated with mitochondrial alterations. MNGIE is characterized by severe gastrointestinal dysmotility, cachexia, ophthalmoplegia, ptosis, peripheral neuropathy, and leukoencephalopathy. The condition is caused by mutation of the TYMP gene. We studied the clinical and biochemical characteristics of a family with MNGIE. The proband was a 48-year-old male presenting with diarrhea and progressive weight loss. He also had ptosis and exhibited eyeball fixation. His blood and cerebrospinal fluid lactate levels were elevated. Magnetic resonance imaging of the brain revealed diffuse leukoencephalopathy. Ragged red fibers and cytochrome c oxidase-deficient fibers were apparent on muscle biopsy. His vision and ptosis deteriorated significantly during follow-up. Our clinical diagnosis of MNGIE was confirmed by TYMP gene analysis. We discovered a homozygous TYMP c.1193-1216 dup-GGGCGCTGCCGCTGGCGCTGGTGC mutation (a duplication). Some of the family members were heterozygous for the mutation but had no clinical features. We predicted the function of this mutation using PredictProtein and found that the secondary structure had changed in the region of the helix and strand, the transmembrane region, and the protein-protein binding sites. The family described herein exhibited biochemically, genetically, and functionally confirmed MNGIE syndrome.

// Ying-Li Lin 1, * , Yan-Ling Wang 2, * , Xing-Li Fu 3 , Wen-Ping Li 4 , Yu-Hao Wang 5 , Jian-Guo Ma 4 1 Department of Urology, Xuzhou Cancer Hospital, Affiliated Xuzhou Hospital of Jiangsu University, Xuzhou, 221000, Jiangsu, China 2 Department of Anesthesiology, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510630, Guangdong, China 3 Health Science Center, Jiangsu University, Zhenjiang, 212000, Jiangsu, China 4 Department of Urology, Third Hospital of Hebei Medical University, Shijiazhuang, 050051, Hebei, China 5 Department of Clinical Medicine, Nanjing Medical University, Nanjing, 210000, Jiangsu, China * These authors contributed equally to this work Correspondence to: Jian-Guo Ma, email: mjg2014sjz@163.com Keywords: bladder cancer, biomarker, protocadherin7, PCDH7, prognosis Received: December 04, 2015 Accepted: February 23, 2016 Published: April 07, 2016 ABSTRACT Bladder cancer is a heterogeneous disease with outcome difficult to predict, and novel predictive biomarkers are needed. PCDH7, a member of protocadherins family, functions as tumor suppressor in several human cancers. The human PCDH7 gene is localized in chromosome 4p15, which is often inactivated in human cancers, including bladder cancer. The aim of this study was to investigate the clinical significance of PCDH7 expression in non-muscle invasive bladder cancer (NMIBC). PCDH7 expression was examined using immunohistochemical staining in 199 primary NMIBC tissues and 25 normal bladder epithelial tissues. Then the relationship between PCDH7 expression and clinicopathologic features was evaluated. Kaplan-Meier survival analysis and Cox analysis was used to evaluate the correlation between PCDH7 expression and prognosis. PCDH7 expression in NMIBC tissues was significantly lower than that in normal bladder epithelial tissues ( P < 0.001). Low PCDH7 expression correlated with advanced grade ( P = 0.021) and larger tumor size ( P = 0.044). Moreover, patients with low PCDH7 expression have shorter recurrence-free survival ( P < 0.001), progression-free survival ( P = 0.007) and overall survival ( P = 0.011) than patients with high PCDH7 expression. Low PCDH7 expression is an independent predictor of recurrence-free survival (multivariate Cox analysis: P = 0.007), progression-free survival (multivariate Cox analysis: P = 0.014) and overall survival (multivariate Cox analysis: P = 0.004). The findings indicate that low PCDH7 expression is a potential prognostic biomarker for primary NMIBC.

The placental syncytiotrophoblast, which is formed by the fusion of cytotrophoblast cells, is indispensable for the establishment and maintenance of normal pregnancy. The human endogenous retrovirus envelope glycoprotein syncytin-2 is the most important player in mediating trophoblast cell-cell fusion as a fusogen. We constructed expression plasmids of wild-type and 21 single-amino-acid substitution mutants of syncytin-2, including 10 N-glycosylation sites individually silenced by mutagenizing N to Q, 1 naturally occurring single-nucleotide polymorphism (SNP) N118S that introduced an N-glycosylation site, and another 10 non-synonymous SNPs located within important functional domains. We observed that syncytin-2 was highly fusogenic and that the mutants had different capacities in merging 293T cells. Of the 21 mutants, N133Q, N312Q, N443Q, C46R (in the CXXC motif) and R417H (in the heptad repeat region and immunosuppressive domain) lost their fusogenicity, whereas N332Q, N118S, T367M (in the fusion peptide), V483I (in the transmembrane domain) and T522M (in the cytoplasmic domain) enhanced the fusogenic activity. We also proved that N133, N146, N177, N220, N241, N247, N312, N332 and N443 were all glycosylated in 293T cells. A co-immunoprecipitation assay showed compromised interaction between mutants N443Q, C46R, T367M, R417H and the receptor MFSD2A, whereas N118S was associated with more receptors. We also sequenced the coding sequence of syncytin-2 in 125 severe pre-eclamptic patients and 272 normal pregnant Chinese women. Surprisingly, only 1 non-synonymous SNP T522M was found and the frequencies of heterozygous carriers were not significantly different. Taken together, our results suggest that N-glycans at residues 133, 312, 332 and 443 of syncytin-2 are required for optimal fusion induction, and that SNPs C46R, N118S, T367M, R417H, V483I and T522M can alter the fusogenic function of syncytin-2.

Pre-eclampsia (PE) is a serious hypertensive disorder of pregnancy that remains a leading cause of perinatal and maternal morbidity and mortality worldwide. Placental ischemia/hypoxia and the secretion of soluble fms-like tyrosine kinase 1 (sFlt1) into maternal circulation are involved in the pathogenesis of PE. Although low-dose aspirin (LDA) has beneficial effects on the prevention of PE, the exact mechanisms of action of LDA, particularly on placental dysfunction, and sFlt1 release, have not been well investigated. This study aimed to determine whether LDA exists the protective effects on placental trophoblast and endothelial functions and prevents PE-associated sFlt1 release. First, we observed that LDA mitigated hypoxia-induced trophoblast apoptosis, showed positive effects on trophoblast cells migration and invasion activity, and increased the tube-forming activity of human umbilical vein endothelial cells (HUVECs). In addition, LDA decreased hypoxia-induced sFlt1 production, and the c-Jun NH2 -terminal kinase/activator protein-1 (JNK/AP-1) pathway was shown to mediate the induction of sFlt1. Moreover, the transcription factor AP-1 was confirmed to regulate the Flt1 gene expression by directly binding to the Flt1 promoter in luciferase assays. The result of chromatin immunoprecipitation assays further demonstrated that LDA could directly decrease the expression of the transcription factor AP-1, and thus decrease sFlt1 production. Finally, the effects of LDA on sFlt1 production were proved in human placental explants. Taken together, our data show the protective effects of LDA against trophoblast and endothelial cell dysfunction and reveal that the LDA-mediated inhibition of sFlt1 via the JNK/AP-1 pathway may be a potential cellular/molecular mechanism for the prevention of PE.

Large-scale quantification of protein O-linked β- N-acetylglucosamine (O-GlcNAc) modification in a site-specific manner remains a key challenge in studying O-GlcNAc biology. Herein, we developed an isotope-tagged cleavable linker (isoTCL) strategy, which enabled isotopic labeling of O-GlcNAc through bioorthogonal conjugation of affinity tags. We demonstrated the application of the isoTCL in mapping and quantification of O-GlcNAcylation sites in HeLa cells. Furthermore, we investigated the O-GlcNAcylation sensitivity to the sugar donor by quantifying the levels of modification under different concentrations of the O-GlcNAc labeling probe in a site-specific manner. In addition, we applied isoTCL to compare the O-GlcNAcylation stoichiometry levels of more than 100 modification sites between placenta samples from male and female mice and confirmed site-specifically that female placenta has a higher O-GlcNAcylation than its male counterpart. The isoTCL platform provides a powerful tool for quantitative profiling of O-GlcNAc modification.

Background Preeclampsia, a serious pregnancy-associated syndrome, is the leading cause of maternal and perinatal morbidity and mortality. Significant exacerbation of the hypercoagulation status as well as imbalanced steroid hormones have been reported in developed preeclampsia. However, it remains unclear whether the two pathological changes are directly associated. Method and results Our proteomic analysis revealed a significantly elevated SerpinF2/α2-antiplasmin level in preeclampsia plasma. Measurement of the longitudinally gestational change of plasmin-α2-antiplasmin (PAP) complex, testosterone, estradiol in preeclampsia patients and normal pregnant women demonstrated that the circulating PAP and testosterone levels in the early-onset preeclampsia (E-PE) patients were substantially higher, whereas estradiol concentration was significantly lower than that in normal pregnant controls from early pregnancy throughout gestation. Correlation analysis revealed that circulating PAP is in positive correlation with the concentration of testosterone, and in negative correlation with estradiol in E-PE patients. In E-PE placenta, the productions and activities of 17β-hydroxysteroid dehydrogenases 3 and aromatase, the essential enzymes for testosterone and estradiol synthesis, were compromised. In human renal and trophoblastic cells, testosterone and estradiol could regulate SerpinF2 expression in opposite ways. In addition, obvious fibrin deposition was colocalized with SerpinF2 in intervillous spaces and the area surrounding syncytiotrophoblasts in E-PE placenta. Conclusion The findings reveal a tight correlation between the imbalanced steroid hormone production and the procoagulation factor in E-PE patients, which provide potential biomarkers to predict preeclampsia, and bring new insight into the pathogenesis of preeclampsia.

Obesity and related metabolic diseases are becoming worldwide epidemics that lead to increased death rates and heavy health care costs. Effective treatment options have not been found yet. Here, based on the observation that baicalin, a flavonoid from the herbal medicine Scutellaria baicalensis, has unique antisteatosis activity, we performed quantitative chemoproteomic profiling and identified carnitine palmitoyltransferase 1 (CPT1), the controlling enzyme for fatty acid oxidation, as the key target of baicalin. The flavonoid directly activated hepatic CPT1 with isoform selectivity to accelerate the lipid influx into mitochondria for oxidation. Chronic treatment of baicalin ameliorated diet-induced obesity (DIO) and hepatic steatosis and led to systemic improvement of other metabolic disorders. Disruption of the predicted binding site of baicalin on CPT1 completely abolished the beneficial effect of the flavonoid. Our discovery of baicalin as an allosteric CPT1 activator opens new opportunities for pharmacological treatment of DIO and associated sequelae.

To investigate the surgical techniques and mid-term efficacy of reconstruction for proximal femur with allograft-prosthetic composite (APC) .Fifteen patients who underwent uncemented APC reconstruction of proximal femur after bone tumor resection were retrospectively evaluated. Image and physical examinations were taken on the 1st,3rd,6th,9th and 12th month after surgery and every 6 months thereafter to assess union condition,greater trochanteric bone absorption and myodynamia of abductors. Musculoskeletal Tumor Society (MSTS) and Harris Hip Score (HHS) were utilized to evaluate the function of hip joint.Seven male and eight female patients were included with average age of 25.1 years(17-56 years) and average followup of 32.8 months(18-48 months). Four patients had giant cell tumor of the bone,3 patients had osteosarcoma,3 patients had chondrosarcoma,2 patients had malignant fibrous histiotoma,2 patients had osteoblastoma and 1 patient had Ewing sarcoma. Unions occurred in the allograft-host bone interfaces of all patients in the period of 5-9 months postoperatively. Bone absorption in the allograft-host bone interfaces,dislocation,hypersensitivity of the allograft and acetabulum abrasion were not observed,while there were no metastasis and tumor recurrence. Three patients had periprosthetic fractures intraoperatively and were well fixed with cerclage. Absorptions in the greater trochanteric happened to 6 patients. All patients had mean MSTS and HHS scores of 11.3 (7-15) and 47.3 (40.3-58.5) preoperatively and 26.1(24-29) and 80.1(66.2-92.7) postoperatively.Uncemented APC reconstruction is proper for young patients with long life expectancy and high demand of function that suffered from tumors of proximal femur.

Gestational diabetes mellitus (GDM) is the primary cause of maternal and fetal morbidity and mortality. Insulin resistance (IR) is pivotal to GDM pathogenesis, and mammalian target of rapamycin (mTOR) is a critical regulator of GDM. An increasing amount of evidence indicates that vitamin D deficiency is a risk factor for GDM. However, there are few reports on the effect of IR on GDM placentas. The present study aims to verify that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) can ameliorate trophoblast IR by suppressing mTOR signaling. An IR BeWo cell model was established in the presence of high insulin and glucose medium. The IR level and mTOR activation with or without 1,25(OH)2D3 treatment were evaluated. The IR cells exhibited excessive mTOR signaling activation, upregulated inflammatory factor levels, and extensive lipid infiltration. However, 1,25(OH)2D3 reversed mTOR activation and reduced the IR level and lipid infiltration. In addition, 1,25(OH)2D3 treatment in GDM placental explants blocked the aberrant, increased levels of leptin, TNF-α, and IL-6. Therefore, 1,25(OH)2D3 treatment protects trophoblasts against high IR mainly through suppressing mTOR signaling, and this mechanism may serve as a potential therapy for patients with GDM.

Proper differentiation of trophoblast cells in the human placenta is a prerequisite for a successful pregnancy, and dysregulation of this process may lead to malignant pregnancy outcomes, such as preeclampsia. Finding specific markers for different types of trophoblast cells is essential for understanding trophoblast differentiation. Here, we report that placenta-specific protein 8 (PLAC8) is specifically expressed in the interstitial extravillous trophoblast cells (iEVTs) on the fetomaternal interface. Using model systems, including placental villi-decidua co-culture, iEVTs induction by using primary trophoblast cells or explants, etc., we found that PLAC8 promotes invasion and migration of iEVTs. Mechanistically, time-lapse imaging, GTPase activity assay, co-immunoprecipitation and RNA-seq studies show that PLAC8 increases the Cdc42 and Rac1 activities, and further induces the formation of filopodia at the leading edge of the migratory trophoblast cells. More interestingly, PLAC8 is significantly upregulated under hypoxia and expression of PLAC8 is higher in iEVTs from preeclamptic placentas when compared with those from the normal control placentas. Together, PLAC8 is a new marker for iEVTs and plays an important role in promoting trophoblast invasion and migration., Highlighted Article: Oxygen tension-dependent expression of placenta-specific protein 8 positively regulates trophoblast invasion and migration partially through upregulating the activation of Rac1 and Cdc42.

To design minimal invasive screw on posterior pelvic ring and perform threedimensional finite element analysis based on a pelvis finite element model.We measured the pelvic anatomical data of 20 healthy volunteers and identified potential designs for minimal invasive screw on posterior pelvic ring. A finite element model of pelvis was then established. Threedimensional finite element analyses were performed under static and dynamic mechanical loading,respectively.Three screw tracks on ilium (A,B and C) were identified based on a threedimensional reconstruction of pelvis. Nail track B and C had greater length and width,but shorter distance between nailing and soft tissue compared with nail track A. Static loading under an external rotation load of 500 N generated a maximum Mises Von stress of 582.05 Pa and sacral iliac complex of 107.38 Pa. The greatest strain was located at the articular cartilage on the side of the nail,followed by lateral sacral joint cartilage and symphysis pubis. The largest displacement was located at the ilium on the side of the nail,with a gradient decrease to the opposite side. The largest displacement of the anterior superior iliac spine was 0.35 cm on the side of the nail. The dynamic loading identified displacement of the anterior superior iliac spine with 1.5 mm inTaking B/C as a main screw track and A as an auxiliary screw track is a reasonable choice. The pelvic finite element model lays a foundation for further studies into sacral fracture and design of screw tracks.

Thyroid-associated ophthalmopathy (TAO) is an autoimmune disease. Studies showed that T helper 1 (Th1), Th2, and Th17 cells play important roles in the pathology of TAO. Tim-3 and its only known ligand Galectin-9 (Gal-9) is related to the suppression of Th1 and Th17 cytokine secretion. This study aims to investigate the role of Tim3/Gal-9 in the inflammatory response of TAO. In this study, the levels of Tim3, Gal-9, and cytokines of Th1 (TNF-α and IFN-γ), Th2 (IL-4), and Th17 (IL-17) cells were analyzed in the blood samples of TAO patients and healthy controls as well as in orbital fibroblasts. Tim3 overexpression and Gal-9 neutralizing antibody were used in TAO and LPS-stimulated control orbital fibroblasts to further investigate the role and mechanism of Tim3/Gal-9 on the inflammation of TAO. We found Tim3 and Gal-9 expression was significantly downregulated in TAO patients and further lower in active TAO than inactive TAO or controls. Th1, Th2, and Th17 cytokines were all increased in TAO patients. Th1 and Th17 cytokines were higher in active TAO patients than in inactive TAO patients, while Th2 cytokines were enhanced in inactive TAO. Tim3 overexpression decreased the levels of Th1 and Th17 cytokines, but not Th2 cytokine in TAO or LPS-stimulated control orbital fibroblasts. These effects were abrogated by Gal-9 neutralizing antibody. Moreover, Tim3 reduced the levels of p-Akt and p-p65 in TAO or LPS-induced control orbital fibroblasts that were reversed by Gal-9 blocking. In conclusion, Tim3/Gal-9 alleviates the inflammation of TAO patients via suppressing Akt/NF-κB signaling pathway.

BACKGROUND PCDH8 is a tumor suppressor that regulates cell adhesin, proliferation, and migration. It is often inactivated by aberrant promoter methylation in several human cancers, including clear cell renal cell carcinoma (CCRCC). The clinical significance of PCDH8 methylation in CCRCC remains unclear. The aim of this study was to investigate the relationship between PCDH8 methylation and clinicopathological characteristics as well as outcome of patients with CCRCC. MATERIAL/METHODS The methylation status of PCDH8 in 153 CCRCC tissues and 97 paired adjacent normal renal tissues were examined using methylation-specific PCR (MSP). Then the relationships between PCDH8 methylation and clinicopathological features as well as progression-free survival of CCRCC patients were evaluated. RESULTS PCDH8 methylation was significantly more frequent in CCRCC tissues compared with normal renal tissues. Moreover, PCDH8 methylation was significantly correlated with advanced clinical stage (P=0.0141), higher grade (P=0.0190), and lymph node metastasis (P=0.0098). In addition, multivariate analysis showed that PCDH8 methylation was independently associated with poor progression-free survival (P=0.0316). CONCLUSIONS PCDH8 methylation is a frequent event in CCRCC and is correlated with unfavorable clinicopathological features. Moreover, PCDH8 methylation may be a useful biomarker to predict the progression of CCRCC.

Preeclampsia is a pregnancy-specific syndrome manifested by the onset of hypertension and proteinuria after the 20th week of gestation. Abnormal placenta development has been generally accepted as the initial cause of the disorder. Recently, microRNA-210 (miR-210) has been found to be upregulated in preeclamptic placentas compared with normal placentas, indicating a possible association of this small molecule with the placental pathology of preeclampsia. However, the function of miR-210 in the development of the placenta remains elusive. The aim of this study was to characterize the molecular mechanism of preeclampsia development by examining the role of miR-210. In this study, miR-210 and potassium channel modulatory factor 1 (KCMF1) expressions were compared in placentas from healthy pregnant individuals and patients with preeclampsia, and the role of miR-210 in trophoblast cell invasion via the downregulation of KCMF1 was investigated in the immortal trophoblast cell line HTR8/SVneo. The levels of KCMF1 were significantly lower in preeclamptic placenta tissues than in gestational week–matched normal placentas, which was inversely correlated with the level of miR-210. KCMF1 was validated as the direct target of miR-210 using real-time polymerase chain reaction, Western blotting, and dual luciferase assay in HTR8/SVneo cells. miR-210 inhibited the invasion of trophoblast cells, and this inhibition was abrogated by the overexpression of KCMF1. The inflammatory factor tumor necrosis factor-α could upregulate miR-210 while suppressing KCMF1 expression in HTR8/SVneo cells. This is the first report on the function of KCMF1 in human placental trophoblast cells, and the data indicate that aberrant miR-210 expression may contribute to the occurrence of preeclampsia by interfering with KCMF1-mediated signaling in the human placenta.

The purpose of this study was to investigate whether common polymorphisms in the C-terminus of SCNN1B gene encoding the β-subunit of epithelial sodium channel are associated with essential hypertension (EH) in Chinese hypertensive families. A total of 433 subjects from 102 EH families were recruited. Biochemical and anthropometric indices and systematic screening of the C-terminus of SCNN1B were performed. Four single nucleotide polymorphisms (SNPs) were found. Homozygotes for the common A allele at rs3743966 had on average a 12.06 mmHg higher SBP and a 7.43 mmHg higher DBP than homozygotes for the rarer T allele. AA + AT genotype of rs3743966 was also found to maybe a risk factor of hypertension by logistic regression and transmission/disequilibrium test. AA + AT genotype of rs3743966 maybe a risk factor of EH. In conclusion, there was a significant association between the rs3743966 SNP in intron 12 and EH in Chinese hypertensive families.

The aim of this study was to better understand methicillin-resistant Staphylococcus aureus (MRSA) at the molecular level by investigating the genotypic characteristics and evolutionary patterns of MRSA clones in Shenyang, China.We analyzed the molecular epidemiology of 60 MRSA isolates in Shenyang, China, between 2002 and 2008, using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and S. aureus protein A (spa) typing. They were examined for their antimicrobial susceptibilities.Among the 60 isolates, ST239 was identified most frequently (34 isolates; 58%), followed by ST5 (20 isolates; 34%). Nine spa types were obtained and 4 PFGE strain families (A, B, C, and D) were resolved. Spa type t030, which corresponded to PFGE genotypes A1, A3, and A4, constituted 45% (27/60) of all isolates; spa type t037, which corresponded to PFGE type A2, accounted for 13% (8/60) of all isolates. These 2 spa genotypes belonged to ST239 and carried SCCmec type III. Isolates genotyped as spa type t002 comprised 27% (16/60) of the study set and included isolates typed as PFGE B1 and B2, ST5, and SCCmec II. Most of MRSA isolates belonging to ST239 were susceptible to trimethoprim-sulfamethoxazole. The minimum inhibitory concentration (MIC50) of vancomycin among MRSA isolates belonging to ST5 (2 mg/l) was higher than that for other isolates (1 mg/l).These data document 2 major epidemic MRSA clones in Shenyang, China: ST239-MRSA-SCCmec type III-t037/t030 and ST5-MRSA-SCCmec type II-t002.

Preeclampsia is a major disorder of pregnancy and a leading cause of maternal and perinatal morbidity and mortality. MicroRNAs are small noncoding RNAs that regulate gene expression posttranscriptionally. In this study, we examined the expression of miR-376c and found that miR-376c levels were downregulated in both placental and plasma samples collected from preeclamptic patients, when compared with the normal pregnant women at the same gestational stage. Overexpression of miR-376c induced trophoblast cell proliferation, migration, and invasion in HTR8/SVneo cells and promoted placental explant outgrowth. In contrast, inhibition of endogenous miR-376c resulted in a decrease in trophoblast cell invasion and placental explant outgrowth. We identified activin receptor-like kinase 5 (ALK5), a type I receptor for transforming growth factor-β, and ALK7, a type I receptor for Nodal, as targets of miR-376c. Overexpression of miR-376c repressed transforming growth factor-β and Nodal functions, whereas overexpression of ALK5 and ALK7 reversed the effects of miR-376c. These results demonstrate that miR-376c inhibits both ALK5 and ALK7 expression to impair transforming growth factor-β/Nodal signaling, leading to increases in cell proliferation and invasion. An unbalanced Nodal/transforming growth factor-β and miR-376c expression may lead to the development of preeclampsia.

Preeclampsia, a multisystem syndrome occurring during mid- to late gestation in humans, is a leading cause of maternal and perinatal morbidity and mortality. Patients usually present with high circulating testosterone and reduced estradiol production, but the mechanisms remain unclear. Revealing the mechanism that modulating the imbalance of testosterone and estradiol in preeclampsia is of great value in understanding the cause of the disease. The placenta is the predominant source of steroid hormone production during gestation, and we observed markedly increased 17β-HSD3 (17β-hydroxysteroid dehydrogenase 3) levels and downregulated aromatase expression, the key enzymes responsible for synthesis of testosterone and estradiol, respectively, in preeclamptic placentas compared with controls. Furthermore, we found a significant upregulation of microRNA (miR)-22 in preeclamptic placentas. In a trophoblast cell line, JEG-3 cells, testosterone repressed the expression of aromatase and estrogen receptor α and the production of estradiol while promoting miR-22 expression. miR-22 directly targeted and inhibited estrogen receptor α expression while indirectly decreasing aromatase expression and estradiol production by interfering with estrogen receptor α signaling. Furthermore, inhibition of miR-22 expression significantly reversed the inhibitory effect of testosterone on de novo estradiol synthesis in human trophoblastic cells. The findings reveal a mechanism underlying the balanced production of androgen and estrogen modulated by miR-22 in the human placenta and provide new insights into the pathogenesis of preeclampsia from the aspect of endocrine regulation.

OBJECTIVE: To investigate the prevalence of adenoviruses (AdV) and their genotypes in infants and young children with diarrhea. METHODS: A total of 380 children with diarrhea aged less than 3 years were enrolled. The genomic DNA was extracted from stool and PCR was used to detect AdV. Clone sequencing and genotyping were performed for DNA in AdV-positive specimens. RESULTS: AdV was detected in 24 out of 380 specimens, and the detection rate was 6.3% (24/380). A majority of children with positive AdV were aged 2-3 years. The viral sequence analysis of positive specimens showed that the detection rates of enteric AdV41 and non-enteric AdV were 4.2% (16/380) and 2.1% (8/380), respectively, and among the children with non-enteric AdV, there were 2 with AdV1, 2 with AdV2, 1 with AdV7, 2 with AdV12, and 1 with AdV31. CONCLUSIONS: Diarrhea caused by AdV is commonly seen in children aged 2-3 years, and AdV41 is the major predominant strain.

Synovial chondromatosis of the temporomandibular joint(TMJ) is an extremely rare condition. A case of synovial chondromatosis with postoperative recurrence and involvement of base of skull was reported. The clinic pathological features, diagnosis and treatment were discussed with literature review.

Objective Preeclampsia is a pregnancy-related syndrome. Shallow invasion of uterine wall by trophoblast cells has been generally accepted as the major pathological change of this disorder. We previously found downregulation of miR-195 in preeclamptic placentas. Bioinformatic analysis predicted a type II activin receptor, activin receptor type-2B (ActRIIB), as one of the potential targets of miR-195. Considering the key function of activin A on trophoblast cell behaviors and placenta development, we proposed miR-195 may affect trophoblast cell invasion by repressing the expression of ActRIIB. Methods The colocalization of ActRIIB and miR-195 in human placenta was measured by in-situ hybridization and immunohistochemistry. Western blotting, real-time PCR and dual luciferase assay were performed in human trophoblast cell line, HTR8/SVneo cells, to validate the targeting of ActRIIB by miR-195. Cell invasiveness was analyzed using transwell insert invasion assay in HTR8/SVneo cells. Results In human placenta, ActRIIB and miR-195 exhibited similar localization in various subtypes of trophoblast cells, including villous and extravillous trophoblasts. The protein expressions of ActRIIB in preeclamptic placenta were significantly higher as compared with the normal controls, which was opposite to the changing pattern of miR-195. In HTR8/SVneo cells, miR-195 could directly target and suppress the expression of ActRIIB. Meanwhile, the invasion-promoting effect of miR-195 on trophoblast cells could be largely impeded by ActRIIB overexpression. Conclusion In human trophoblast cells, miR-195 could promote cell invasion via directly targeting ActRIIB. The impaired miR-195 expression may contribute to the occurrence or development of preeclampsia through interfering with activin/nodal signaling in the placenta.

Objective To investigate whether more benefits can be achieved through high intensity atorvastatin compared with moderate intensity atorvastatin in acute coronary syndrome (ACS) patients with diabetes mellitus (DM). Methods This was a randomized controlled trail. Total 591 ACS patients with DM who underwent percutaneous coronary intervention were enrolled, 297 in high intensity atorvastatin group (40mg/day) and 294 in moderate intensity atorvastatin group (20mg/day). The primary end point was one-year incidence of major adverse cardiovascular events (MACE, including cardiovascular death, spontaneous myocardial infarction, unplanned revascularization). Cox proportional hazard regression models were used to analyze the association between clinical endpoints and atorvastatin treatment. Results At the end of one-year, low-density lipoprotein cholesterol level was lower in high intensity group than in moderate group (1.6±0.6 vs 1.8±0.6, p=0.041). MACE in high intensity group decreased 44.5% than moderate group (8.4% vs. 14.6%, p=0.018). The adjusted hazard ratio (HR) for MACE in patients with atorvastatin 40mg/d was lower compared to patients with atorvastatin 20mg/d (HR [95% CI] 0.61 [0.36 to 0.91], p=0.026). The rates of adverse events were no significantly different between the two groups. Conclusions For ACS patients with DM, high intensity atorvastatin induced better long-term outcomes compared with moderate intensity.

The present cross-sectional study was conducted to assess and compare the prevalence of dental caries of 229 deaf adolescents in a special senior high school and to identify factors related to dental caries, with a match group of 196 healthy adolescents in a normal senior high school, in Jiangsu province of East China. In this study the prevalence of dental caries was 55.9%, Decayed, Missing and Filled Teeth (DMFT) index in deaf students was 1.40±1.89; in healthy students was 13.8% and 1.36±1.72, respectively. Relatively lower proportion of awareness on dental health knowledge and lower proper practice of dental health behavior have been observed among deaf students compared with the control group. Multiple logistic regressions showed that the risk factors of dental caries in deaf students involved high frequency of consuming dessert and carbonated beverage. Poor oral health, lack of oral health knowledge and proper oral health practice could be found in deaf students. The dental treatment for the two groups was urgently needed. The dental health education concerning dietary behavior and prevention program to the deaf students and their parents should be reinforced; the supervision of oral health behavior for deaf and healthy students needs to be strengthened.

Preeclampsia is a pregnancy-specific syndrome mainly characterized by hypertensive disorder and proteinuria after gestational weeks 20. So far the etiology of preeclampsia remains unclear. We previously reported that preeclamptic placentas exhibited decreased mRNA expression and hypermethylation in promoter region of the paternally imprinted H19 gene compared with normal placentas. H19 has recently been identified to encode the precursor of miR-675, indicating a possible novel functional pathway of the imprinting gene. The aim of the present study was to identify the roles of H19 gene via miR-675 pathway in human trophoblast cells, and to figure out the involvement of this pathway in pathogenesis of preeclampsia. Knockdown of H19 gene or inhibition of miR-675 exhibited similar proliferation-promoting effect in human trophoblastic JEG-3 cells. Target gene prediction in combination with luciferase assay revealed that miR-675 could directly downregulate Nodal Modulator 1 (NOMO1) protein expression by binding to 3'-UTR sequence of NOMO1. Overexpression of NOMO1 in JEG-3 cells could rescue miR-675-surppressed cell proliferation and phosphorylation of Smad2, while Nodal had additive effect with miR-675 in suppression cell proliferation and activation of Smad2. In early-onset preeclamptic placentas, expression levels of H19 gene and miR-675 were appreciably lower, while NOMO1 protein level was higher than those in normal placentas. Taken together, our data suggested that H19 gene could inhibit human trophoblast cell proliferation via encoding miR-675 that targeted NOMO1, and aberrantly lowered expression of H19 in placenta may participate in the excessive proliferation of trophoblast cells observed in early-onset severe preeclampsia by downregulating miR-675 which targets NOMO1 and interferes with Nodal signaling.

Progesterone has been used in the hormonal treatment of endometrial cancer (EC) for many years, but the response rates are unsatisfying. The down-regulated progesterone receptor (PR) is the main reason for treatment failure. The insulin-like growth factor (IGF) system is related to EC risk, and IGF-I can inhibit PR transcription in breast cancer. Recent evidence suggests that metformin-combined oral contraceptives may reverse progesterone-resistant atypical endometrial hyperplasia, but the mechanism is unclear. We attempt to investigate the interaction of metformin, PR and IGF-II expression, and identify whether metformin can enhance the antitumor effect of medroxyprogesterone acetate (MPA) using Ishikawa and HEC-1B EC cell lines. We found that both IGF-I and IGF-II inhibit PR A/B mRNA and protein expression, whereas metformin markedly promotes PR expression. In parallel, IGF-II increases phosphorylation of AKT and p70S6K, while metformin increases AMPK phosphorylation and decreases p70S6K phosphorylation. The effects of metformin on PR A/B and p70S6K are partially reversed by an AMPK inhibitor. Furthermore, metformin synergistically antiproliferates MPA in two cell lines, with the peak synergy occurring with 10μM metformin combined with 1μM MPA (CI=0.20448 for Ishikawa, CI=0.12801 for HEC-1B). Our results demonstrate that metformin promotes PR expression, which can be inhibited by overexpressed IGF-II in EC. This effect is partially mediated through activating AMPK followed by inhibiting the overactivated mTOR pathway.

Preeclampsia (PE) is a severe hypertensive disorder associated with pregnancy; despite substantial research effort in the past several years, the etiology of PE is still unclear. The role of epigenetic factors in the etiology of PE, including DNA methylation, has been poorly characterized. In the present study, we investigated global DNA methylation as well as DNA methylation of the paternally imprinted H19 gene in preeclamptic placentas. Using 5-methylcytosine immunohistochemistry and Alu and LINE-1 repeat pyrosequencing, we found that the global DNA methylation level and the DNA (cytosine-5) methyltransferase 1 mRNA level were significantly higher in the early-onset preeclamptic placentas when compared with the normal controls. Data from methylation-sensitive high resolution melting demonstrated hypermethylation of the promoter region of the H19 gene, and results of real-time PCR showed decreased mRNA expression of H19 gene in the early-onset preeclamptic placentas as compared with the normal controls. Our results suggest that abnormal DNA methylation during placentation might be involved in the pathophysiology of PE, especially early-onset preeclampsia.

Objective This study was undertaken to determine the relationship between red cell distribution width (RDW) and short-term outcomes in acute coronary syndrome (ACS) in a Chinese population. Methods and Patients A total of 1,654 patients with ACS on admission were divided into four groups according to quartiles of baseline RDW. The relationships between RDW and one-month cardiac mortality as well as heart failure and recurrent infarction were assessed. Results Higher RDW values were associated with increased one-month cardiac mortality (quartile 1: 0.2%; quartile 2: 0.6%; quartile 3: 3.3%; quartile 4: 8.1%; p